Single cell analysis (SCA) is an active area of research and commercial development for determining cellular heterogeneity and monitoring cellular functions. Single cell applications focus on classification of subpopulation based on immunophenotyping or sequencing of genomic content. Clinical applications of SCA include the screening for rare cells, for example circulating tumor cells (CTCs), in an enriched sample.
Current methods for cell isolation include limiting dilution using large liquid handler instruments, flow cytometry, and laser capture microdissection (LCM). Flow cytometry is a high throughput sorting method but is typically limited to single time point analysis, large downstream volumes, and high shear flow that can lower cell viability and limit to certain cell types. Limiting dilution and LCM are labor intensive and in the case of LCM low throughput. Microfluidic platforms are addressing the automation need for single cell isolation and sample preparation for gene expression studies by real-time PCR or sequencing but are typically limited to 100 cells or less. Droplet based microfluidics is capable of addressing both automation and high throughput needs but sequential reagent addition is typically not possible after encapsulation and downstream sorting may be required. Microwell arrays have also been used for isolation of single cells. However, microwell array platforms typically rely on manual addition of reagents and use of a micropipette for retrieval of contents from microwell arrays.
In addition, an alternative method to quantitative polymerase chain reaction (qPCR) is digital PCR. In digital PCR, the reaction mixture is compartmentalized in microchambers such as microwells or in monodisperse droplets (droplet digital PCR). The advantages of digital PCR over qPCR include precision and sensitivity. Although there are several commercialized digital PCR instruments currently available that are based on droplets or microchambers, current platforms typically are not fully integrated or typically lack the capability to retrieve the positive samples for downstream processing.
A need exists for innovative technologies that can integrate and automate single cell isolation, screening, and retrieval at increased throughput in a single experiment. A further need exists for a digital PCR platform with the integrated capability to retrieve positive samples for downstream processing.